Transcription factor SPZ1 promotes TWIST-mediated epithelial-mesenchymal transition and oncogenesis in human liver cancer

Abstract

The epithelial-mesenchymal transition (EMT) is an important process in the progression of cancer. However, its occurrence and mechanism of regulation are not fully understood. We propose a regulatory pathway in which spermatogenic leucine zipper 1 (SPZ1) promotes EMT through its transactivating ability in increasing TWIST1 expression. We compared the expression of SPZ1 and TWIST1 in specimens of hepatocarcinoma cells (HCCs) and non-HCCs. Expression of SPZ1 exhibited a tumor-specific expression pattern and a high correlation with patients' survival time, tumor size, tumor number and progression stage. Moreover, forced expression and knockdown of SPZ1 in hepatoma cells showed that SPZ1 was able to regulate the cellular proliferation, invasion, and tumorigenic activity in a TWIST1-dependent manner in vitro and in vivo. These data demonstrate that SPZ1, a newly dscribed molecule, transactivates TWIST1 promoters, and that this SPZ1-TWIST axis mediates EMT signaling and exerts significant regulatory effects on tumor oncogenesis.

Figure 1

Correlation of SPZ1 with cell proliferation and poor prognosis in HCC tumor patients. (a) Expression of SPZ1 mRNA was compared between HCC samples (n=291) and non-HCC samples (n=243). (b) Expression of SPZ1 protein in HCC samples. Brown, SPZ1 expression; asterisk, area of healthy hepatocytes; blue arrows, SPZ1 expression in cytoplasm; red arrows, SPZ1 expression in nuclei. (c) Kaplan–Meier survival analysis of SPZ1. Red, patients with higher expression (n=83; >3.5-fold than the control region) of SPZ1; black, patients with lower expression (n=208; <3.5-fold than the control region) of SPZ1. (d) Endogenous expression of SPZ1 and TWIST1 in various hepatoma cells. (e) SK-Hep1 with high SPZ1 expression showing greater proliferative activity. (f) Effect of forced and knockdown expression of SPZ1 on cyclin D1, E2F1, Ki67 and ERK1/2 mRNAs. (g) Effect of ectopic and knockdown expression of SPZ1 on cyclin D1, E2F1, Ki67 and ERK1/2 proteins. (h) Serum-starved Hep G2 transformed cells (5 × 10⁵ cells) with pFlag-SPZ1, pFlag, pLKO and SPZ1 shRNAi1 were cultured in DMEM plus 10% FBS for 24 h, stained with BrdU-specific antibody and propidium iodide (PI) and subjected to Fluorescence-Activated Cell Sorting (FACS) analysis to determine the percentage of cells in each phase of the cell cycle. FL1-BrdU, bromodeoxyuridine stained cells; FL2-PI, PI stained cells. (i) Percentages of cells that were stained with BrdU-specific antibodies. The percentage of each cell cycle was measured. Means±s.d. of results of three experiments are shown. a and b, P<0.001.

Figure 2

Ectopic SPZ1 expression promotes proliferation, transformation and tumor growth. (a) Effect of forced expression (SPZ1-GFP) and knockdown of SPZ1 (SPZ1 shRNAi1) on cell growth was measured at 12 h intervals. Expression of SPZ1 and exogenous SPZ1-GFP in each transformant were measured by western blotting (inside Figures). (b) Effect of forced expression (SPZ1-GFP) and knockdown of SPZ1 (SPZ1 shRNAi1) on cell proliferation was measured by BrdU incorporation into SK-Hep1 and Huh 7 cells. (c) Effect of forced and knockdown of SPZ1 on colony formation was measured in SK-Hep1 and Huh 7 cells. (d) Effect of forced and knockdown of SPZ1 on tumor growth of transformed SK-Hep1 or Huh 7 in nude mice (n=6). Internal Figures; the cancer of xenografts of pEGFP and SPZ1 shRNAi1. (e) Tumor was detected in each organ of 6- to 8-month-old SPZ1 transgenic mice prepared in the Material and methods section. Eighteen out of 60 transgenic mice generated the cancer features (n=60). Number in parentheses indicate the number of the mice with cancer characteristics. a and b, P<0.001.

Figure 3

SPZ1 activates the TWIST1 promoter through its G-box. (a) Forced SPZ1 (SPZ1-GFP) activated EMT and stem cell-like marker expression in Hep G2 and Hep 3B cells. Arrow: position of indicated protein. (b) The expression of SPZ1, TWIST1 mRNA was examined in Tet-ON inducible SPZ1-GFP transformants until 8 h after addition of DOX. P<0.001. (c) Western blotting of induced SPZ1 and TWIST until 8 h after induction with DOX is shown. (d) Schematic presentation of a series of luciferase deletions of TWIST1 promoter. Relative luciferase activity in SK-Hep1 was calculated as described in the Materials and methods section. P<0.001. (e) Relative DNA-binding activities in SK-Hep1 were measured as described in the Materials and Methods section. P<0.001. (f) The transactivation activity of SPZ1 on TWIST1 promoter was determined as described in the Materials and Methods section. a, P<0.001. (g) ChIP assay of the TWIST1 promoter with SPZ1 antibodies in various hepatoma lines as described in the Materials and Methods section. a, P<0.001. # indicates cell lines with a lower expression of SPZ1.

Figure 4

SPZ1 expression is correlated with liver cancer development. (a) Spz1 was colocalized with Twist1 in liver tumors from Spz1 transgenic mice. Spz1 or mouse IgG, green; Twist1 or rabbit IgG, red. (b) TWIST1 mRNA was overexpressed in the high SPZ1 group compared with the low SPZ1 group. (c) The expression of SPZ1 mRNA in HCC samples correlated with the TWIST1 mRNA expression. (d) Expression of the SPZ1 and TWIST1 proteins in tumor and normal parts from HCC patients. (e) SPZ1 is colocalized with TWIST1 in tumor parts of HCC patients. Green, SPZ1 expression or mouse IgG; red, TWIST1 expression of rabbit IgG; blue; DAPI expression; white, merged. N, nontumor parts; T, tumor parts in a specimen from a patient with HCC.

Figure 5

Coexpression of SPZ1 and TWIST1 transgenes promotes EMT and invasive activity. (a) Effect of forced coexpression of SPZ1 and TWIST1 introduced into Hep G2 and Hep 3B cells on expression of mesenchymal markers. a, P <0.001 and b, P<0.01. (b) Effect of combination of knockdown siRNA constructs of SPZ1 and TWIST1 introduced into SK-Hep1 cells on expression of mesenchymal markers. Upper panel, mRNA levels; lower panel, protein levels. a, P<0.001 and b, P<0.01. (c) Effect of TWIST1 siRNA on Tet-ON inducible SPZ1-GFP on expression of mesenchymal marker proteins in SK-Hep1 and Huh 7 cells. (d) Analysis of wound healing in Huh 7 transfected with SPZ1 and/or TWIST1 expression vectors as described in the Materials and methods section. (e) Coexpression of SPZ1-GFP and TWIST1 promoted invasive activity in Huh 7. Upper panels, hematoxin staining; lower panels, relative activity of invasion activity. (f) Analysis of wound healing in SPZ1 or TWIST1 shRNAi1-transfected SK-Hep1, as described in the Materials and methods section. (g) SK-Hep1 with SPZ1 and TWIST1 knockdown decreased invasive activity compared with vector-transfected cells. (h) SPZ1 shRNAi1- and TWIST1 shRNAi-transfected SK-Hep1 cells abolished the invasive activity in vivo (n=5). Upper panel, images of inoculated recombinant tumor cells; lower panel, comparison of the relative photon flux activity. All except (a) and (b) are indicated as a and b, P<0.001.